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Field evidence of the effects of the epigeic earthworm Dendrobaena octaedra on the microfungal community in pine forest floor
Soil Biology & Biochemistry 32 (2000) 351±360
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Field evidence of the eects of the epigeic earthworm Dendrobaena octaedra on the microfungal community in pine forest ¯oor M.A. McLean a,*, D. Parkinson b a
Louis Calder Center, Fordham University, 53 Whippoorwill Rd., Armonk, NY 10504, USA Department of Biological Sciences, University of Calgary, Calgary, Alta., Canada T2N 1N4
b
Accepted 6 September 1999
Abstract The eects of the invasion of the epigeic earthworm Dendrobaena octaedra on the forest ¯oor microfungal community were studied in a 90 yr old lodgepole pine forest over 2 yr. Fungi were isolated from the L and FH layers and the Ah and Bm horizons 1 and 2 yr after the introduction of earthworms to plots. High density and biomass of D. octaedra correlated positively with fungal dominance and negatively with fungal richness and diversity in the FH layer and the Ah and Bm horizons. High worm density and biomass dierentiated the fungal communities in the FH layer from those in the L layer and Bm horizons and increased the similarity between the fungal communities in the FH layer and the Ah horizon. Earthworm activities appeared to favour the presence of faster growing fungal taxa. # 2000 Elsevier Science Ltd. All rights reserved. Keywords: Fungal community; Earthworm; Dendrobaena octaedra; Soil fungi; Litter fungi
1. Introduction It is well known that earthworms, through their channelling and mixing of organic matter and mineral soil and comminution of organic matter, have signi®cant eects on soil structure and soil chemical properties and thus on microbial activity and on microbial populations (Brown, 1995; Edwards and Bohlen, 1996; Doube and Brown, 1998). Much less is known of the eects of earthworms on microbial communities (Parkinson and McLean, 1998), although we do know that earthworm casts are a favourable environment for fungal growth (e.g. Brown, 1995; Edwards and Bohlen, 1996; Doube and Brown, 1998), burial of leaf litter by anecic earthworms can signi®cantly reduce phytopathogenic fungal propagules (Niklas and Kennel,
* Corresponding author. Tel.: +1-914-273-3078 x18; fax: +1-914273-2167. E-mail address: [email protected] (M.A. McLean).
1981) and that earthworms can graze selectively on fungi (e.g. Cooke, 1983; Moody et al., 1995). A recent invasion of the epigeic earthworm, Dendrobaena octaedra (Savigny) into lodgepole pine forest ¯oors in southwest Alberta, Canada, has provided the opportunity to investigate the eects of an epigeic earthworm on microbial activity and the fungal community. Two approaches were used in our investigation: short-term (6 months) laboratory studies (McLean and Parkinson, 1997a, 1998) and longer-term (2 yr) ®eld studies (the present experiment; McLean and Parkinson, 1997b). In this soil the activities of D. octaedra have signi®cantly altered the organic layers and upper mineral horizons (McLean and Parkinson, 1997a). These physical changes were accompanied by decreased microbial biomass and fungal-to-bacterial ratio in laboratory studies (Scheu and Parkinson, 1994a; McLean and Parkinson, 1997a), and decreased basal respiration and metabolic quotient in the ®eld (McLean and Parkinson, 1997b). In laboratory mesocosms, the activities of D. octaedra increased fungal
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M.A. McLean, D. Parkinson / Soil Biology & Biochemistry 32 (2000) 351±360
species richness, diversity and number of isolates per particle in the short term (6 months) (McLean and Parkinson, 1998). This was attributed to increased spatial heterogeneity, through the addition of casts and other products of earthworm activities to the types of organic substrates already present in the soil. The organic layers in mesocosms containing high numbers of earthworms were completely homogenized (i.e. the organic layers were composed solely of casts). We hypothesize that the longer term (2 yr) eects of these worms will be reduced spatial heterogeneity and therefore decreased fungal species richness and diversity. Knowing that D. octaedra attains maximum growth in the FH layer and Ah horizon (McLean et al., 1996), and that the physical eects of its mixing activities are strongest in these horizons, we hypothesize that its eects on the fungal community will be most intense in these horizons. We have investigated the eects of D. octaedra on the fungal community in the ®eld over 2 yr, in the context of the previous hypotheses.
2. Materials and methods 2.1. Site description This experiment was conducted in a 90 yr old lodgepole pine (Pinus contorta Loud. var. latifolia Engelm.) forest ¯oor in the Kananaskis Valley in the Rocky Mountains of southwestern Alberta, Canada. For a more detailed description see McLean and Parkinson (1997b). 2.2. Experimental design Five pairs of plots 1 by 2 m were set up in August 1993 in a part of the pine forest which surveys had shown to be free of earthworms. Within each pair of plots, two treatments (worms added, no worms added) were randomly assigned. To each of the worm-treated plots 250 immature and 70 mature specimens mÿ2 of Dendrobaena octaedra were added, with a total biomass of 3.3 g d.w. mÿ2. Plots were sampled in September 1994 and September 1995 for assessment of worm abundance and biomass and the occurrence of fungal taxa. The F and H layers were not separated in this experiment since in all cases worm activities had mixed them together. An Ah horizon had developed in one of the plots at 1 yr and in both plots at 2 yr in the high worm treatment and had not formed in the low worm treatment plots at either time.
2.3. Earthworm abundance and biomass Earthworms were heat extracted (Kempson et al., 1963) from 10.5 cm dia cores taken from each plot at each sampling time, and classi®ed as small immatures (<1 cm length), large immatures, matures (clitellate) and aclitellate adults. Oven dry weights of each size class were used to estimate earthworm biomass. Mean biomass of a mature worm was 27 mg d.w. Worm biomass (abundance) in the high worm plots were 13.6 (462) and 27.7 g d.w. (1385) mÿ2 in 1994 and 39.9 (3233) and 19.6 g d.w. (3349) mÿ2 in 1995. No worms were recorded in the low worm plots in September 1994 and September 1995, although a few worms were observed in these plots during the summer. 2.4. Fungi Three subsamples from each replicate of each plot were collected at both sampling times for assessment of the fungal community. These subsamples were separated into layers/horizons (L, FH, Ah (present only in the high worm treatment) and Bm) and then bulked to form composite samples of each layer/horizon from which fungi were isolated using a washing procedure (Parkinson, 1994). Fifty particles (<0.2 mm) were plated, one per plate, from each replicate layer/horizon. Of these, 30 were plated on 2% malt extract agar plus antibiotics (0.1% streptomycin and 0.05% aureomycin) and 20 were plated on malt extract agar plus antibiotics with 5 mg benomyl lÿ1 to permit the isolation of basidiomycetes (Worrall, 1991). Since the potential number of plates and of fungal isolates would have been very large and dicult to handle if all the plots had been sampled, only the fungal communities in the two plots with the highest and lowest worm biomasses at each sampling time were investigated. Although surveys had shown the area to be free of worms, worms were already invading the forest during the experiment. The September sampling in both years did not reveal the presence of any worms in the low worm biomass plots but a few worms and casts were observed in these plots in both years. Therefore the two treatments will be referred to as the high worm and low worm treatments. 2.5. Statistical analysis Fungal community characteristics (similarity, number of fungal isolates per particle, species richness, dominance (d ) and diversity (1/D )) were calculated from the percent frequency of occurrence data (Magurran, 1988). Number of fungal isolates per particle was the mean number of taxa isolated per particle in each replicate of each treatment. Data were analysed in two ways: using ANCOVA
M.A. McLean, D. Parkinson / Soil Biology & Biochemistry 32 (2000) 351±360 Table 1 Morisita±Horn quantitative similarity index for the fungal communities 1 and 2 yr after the introduction of worms to the ®eld plots 1 yr
2 yr
Low vs. high worms L FH Bm
0.751 0.817 0.754
0.612 0.872 0.486
Low worms L vs. FH FH vs. Bm
0.439 0.540
0.573 0.383
High worms L vs. FH FH vs. Ah FH vs. Bm Ah vs. Bm
0.196 0.495 0.492 0.528
0.099 0.854 0.240 0.312
with ®nal worm biomass as the covariate to take into account both the initial treatment applied and the dierences in ®nal worm biomass; and using principal components analysis (PCA) followed by correlation of the extracted axes with environmental variables (initial treatment, ®nal worm biomass, organic matter content, moisture content, pH, C-to-N ratio). Since the environmental constraints and worm activities were dierent in the dierent layers and horizons sampled, the PCA analysis was conducted on each layer or horizon separately. 3. Results 3.1. Eects on fungal community structure A total of 143 taxa were isolated in the whole study. The fungal communities in the L layer and the Bm horizon in both worm treatments were less similar in yr 2 while those in the FH were more similar (Table 1). The fungal communities in the L and FH layers in the low worm treatment were more similar in yr 2 and those in the FH and Bm horizon were less similar (Table 1). In the high worm treatment, the fungal communities in the L and FH layers were very dierent at both sampling times. In the high worm treatment the fungal communities in the FH and the Ah were more similar in yr 2 while those in the FH and the Ah were less similar to that in the Bm horizon. In the low worm treatment the fungal communities in the L and FH layers and the FH layer and Bm horizon were more similar than those in the same layers at high worm biomass. None of the community characteristics (number of fungal isolates per particle, richness, dominance (d ), diversity (1/D )) calculated were aected by treatment or ®nal worm biomass in either year (Table 2). In the L layer neither ®nal worm biomass or initial
353
treatment aected the fungal community properties. The C-to-N ratio ( p < 0.05) correlated with the ®rst PCA axis which accounted for 99% of the variation in the fungal community data (data not shown). In this layer the C-to-N ratio correlated positively with fungal dominance and negatively with diversity and richness. In the FH layer, the high worm treatment ( p < 0.05) and C-to-N ratio ( p < 0.05) correlated with the ®rst PCA axis which accounted for 98% of the variation in the fungal community characteristics (data not shown). The high worm treatment positively correlated with fungal dominance and negatively with diversity and richness, while the C-to-N ratio correlated positively with diversity and richness and negatively with dominance. In the Ah and Bm horizons, ®nal worm biomass correlated ( p < 0.05) with the ®rst PCA axis, accounting for 97 and 99% of the variation in fungal community data, respectively (data not shown). In both horizons, ®nal worm biomass was positively correlated with fungal dominance and negatively correlated with fungal diversity. In the Ah horizon, ®nal worm biomass was also negatively correlated with fungal species richness. 3.2. Eects on fungal species abundance At 1 yr Trichoderma koningii, Penicillium lanosum, Oidiodendron griseum and basidiomycete 955 occurred more frequently in all layers/horizons in the high worm treatment than in the low worm treatment (Table 3). Percent frequency of occurrence of P. lanosum, O. griseum, Mortierella ramanniana var. ramanniana and M. ramanniana var. angulispora declined in the FH layer with increasing ®nal worm biomass. Percent frequency of occurrence of T. koningii and M. ramanniana var. angulispora increased in the Bm horizon with increasing ®nal worm biomass. Percent frequency of occurrence of basidiomycete 955 increased in the FH layer and decreased in the L layer with increasing ®nal worm biomass. At 2 yr sterile dark 880 occurred more frequently and sterile yellow 842 occurred less frequently in the low than in the high worm treatment (Table 4). None of the taxa were aected by ®nal worm biomass. In the L layer, none of the environmental variables, including initial treatment and ®nal worm biomass, correlated signi®cantly with the PCA axes (data not shown). In the FH layer, the high worm treatment (P < 0.05) and ®nal worm biomass (P < 0.05) correlated with the ®rst PCA axis, accounting for 37% of the variation in the fungal species data (Fig. 1). Trichoderma koningii, Penicillium montanense, basidiomycete 955 and sterile darks 876, 877 and 893 were positively and Mucor an. plumbeus, Mortierella 860 and 864, sterile hyaline 926 and sterile dark 880 were negatively
2.3 (0.4) 1.7 (0.0)
33 (1) 25 (2)
7.4 (0.3) 6.8 (0.1)
0.11 (0.03) 0.15 (0.01)
30.6 (10.6) 18.8 (0.7)
Stot Low High
S8 Low High
d Low High
1/D Low High 14.4 (4.4) 11.5 (0.9)
0.20 (0.03) 0.22 (0.04)
6.6 (0.4) 6.5 (0.0)
32 (5) 28 (2)
3.1 (0.2) 3.0 (0.2)
ND 11.6 (ÿ)
ND 0.24 (ÿ)
ND 6.3 (ÿ)
ND 33 (ÿ)
ND 3.5 (ÿ)
25.1 (10.5) 20.9 (5.7)
0.13 (0.03) 0.15 (0.05)
7.0 (0.6) 6.9 (0.3)
31 (5) 25 (8)
2.6 (0.5) 2.2 (1.1)
Bm
21.9 (0.0) 29.2 (8.0)
0.16 (0.02) 0.05 (0.04)
7.5 (0.1) 7.3 (0.2)
25 (2) 30 (2)
1.8 (0.1) 1.8 (0.1)
L
Ah
L
FH
2 yr
1 yr
Isolates Low High
Treatment
11.6 (2.0) 9.4 (1.4)
0.20 (0.04) 0.27 (0.05)
6.2 (0.3) 6.1 (0.1)
22 (2) 22 (4)
2.8 (0.3) 2.4 (0.4)
FH
ND 10.8 (2.0)
ND 0.23 (0.04)
ND 6.3 (0.3)
ND 28 (5)
ND 2.6 (0.4)
Ah
20.7 (0.9) 31.1 (ÿ)
0.15 (0.01) 0.12 (0.01)
6.9 (0.0) 7.8 (0.2)
14 (2) 18 (10)
0.8 (0.2) 0.9 (0.6)
Bm
Table 2 Mean (standard error) number of fungal isolates per particle plated, fungal species richness (Stot: total number of species, S8: number of species based on sample size of 8 from rarefaction), fungal dominance (d ) and fungal diversity (1/D ) in the L and FH layers and the Ah and Bm horizons 1 and 2 yr after the introduction of Dendrobaena octaedra into ®eld plots n 2). ND: not determined; the Ah horizon was not present in the low worm treatment. Neither initial treatment nor ®nal worm biomass aected the fungal community parameters (P > 0.95)
354 M.A. McLean, D. Parkinson / Soil Biology & Biochemistry 32 (2000) 351±360
b
a
Lower case letters refer to dierences between layers/horizons (P < 0.05). Signi®cant treatment eects across all horizons (P < 0.05).
Trichoderma koningii Oudem. T. polysporum (Link: Fr.) Rifai Penicillium montanense Christensen and Backus P. lanosum Westling Mortierella ramanniana (MoÈller) Linnem. var. ramanniana M. ramanniana var. angulispora (Naumov) Linnem. M. vinacea Dixon-Stewart M. parvispora Linnem. M. humilis Linnem. Oidiodendron griseum Robak Geomyces pannorum (Link) Sigler and Carmichael var. vinaceus (Dal Vesco) van Oorshot Tolypocladium niveum (Rostrup) Bissett Sterile dark 875 Sterile dark 880 Sterile dark 889 Basidiomycete 955
Taxon
2 (2) 20 (17)a 0 (0) 0 (0) 2 (2)a 0 (0) 0 (0) 5 (5) 5 (0) 0 (0) 0 (0) 3 (3) 18 (2)a 7 (0) 11 (4)a 6 (1)a
2 (2) 63 (13)b 2 (2) 0 (0) 35 (0)b 3 (3) 3 (3) 23 (8) 35 (15) 3 (0) 3 (0) 13 (3) 2 (2)b 8 (2) 0 (0)b 0 (0)b
ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND
0 (0) 23 (3)a 27 (17) 2 (2) 7 (3)a 0 (0) 28 (22) 13 (7) 9 (6) 3 (3) 3 (0) 8 (8) 0 (0)b 3 (0) 0 (0)b 0 (0)
Bm
0 (0) 7 (3)a 0 (0) 0 (0)a 0 (0)a 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)a 2 (2) 0 (0) 20 (0)a 0 (0) 10 (7)a 10 (10)a ÿ
L
Ah
L
FH
High worms
Low worms
Ah 27 40 20 3 17 3 5 7 25 3 13 85 0 0 0 0
FH 7 (0)aa 63 (10)b 13 (0) 18 (5)b ÿ 40 (15)b ÿ 7 (7) ÿ 15 (0) 8 (8) 8 (3) 22 (15)b ÿ 2 (2) 3 (3) 3 (3)b 2 (2)a 0 (0)b 5 (5)b +
(ÿ) (ÿ) (ÿ) (ÿ) (ÿ) (ÿ) (ÿ) (ÿ) (ÿ) (ÿ) (ÿ) (ÿ) (ÿ) (ÿ) (ÿ) (ÿ)
17 (17)b +b 10 (10)a 10 (10) 2 (2)ab 5 (5)a 10 (10) + 18 (8) 10 (0) 10 (0) 2 (2)ab 5 (5) 8 (8) 0 (0)b 10 (3)b 0 (0)b 0 (0)b
Bm
Table 3 Mean (standard error) percent frequency of occurrence of most abundant fungal taxa in the L and FH layers and the Ah and Bm horizons 1 yr after the introduction of worms to the ®eld plots n 2). ND: not determined; the Ah horizon was not present in the low worm treatment; only one of the plots in the high worm treatment developed an Ah horizon. +, ÿ refer to positive and negative eects of increasing ®nal worm biomass within horizons across treatments (P = 0.05)
M.A. McLean, D. Parkinson / Soil Biology & Biochemistry 32 (2000) 351±360 355
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M.A. McLean, D. Parkinson / Soil Biology & Biochemistry 32 (2000) 351±360
Table 4 Mean (standard error) percent frequency of occurrence of most abundant fungal taxa in the L and FH layers and the Ah and Bm horizons 2 yr after the introduction of worms to the ®eld plots n 2). ND: not determined; the Ah horizon was not present in the low worm treatment Taxon
Trichoderma polysporum Mortierella ramanniana var. ramanniana M. ramanniana var. angulispora M. vinacea M. parvispora M. humilis Verticillium fungicola var. fungicola Oidiodendron griseum Tolypocladium niveum Sterile dark 875 Sterile dark 880 Sterile dark 919 Sterile yellow 842 a b
Low worms
High worms
L
FH
Ah
Bm
L
FH
Ah
Bm
28 (2)aa 0 (0) 3 (3)a 0 (0) 5 (5)a 5 (0) 0 (0)a 0 (0) 4 (1) 12 (2)a 5 (2) 12 (2)a 13 (3)a
58 (18)b 18 (3) 10 (0)b 8 (2) 13 (3)b 43 (8) 28 (8)b 2 (2) 17 (13) 2 (2)b 10 (3) 0 (0)b 0 (0)b
ND ND ND ND ND ND ND ND ND ND ND ND ND
0 (0)a 5 (5) 3 (3)a 5 (5) 5 (5)a 8 (3) 0 (0)a 0 (0) 0 (0) 0 (0)b 3 (0) 0 (0)b 0 (0)b
3 (3)a 0 (0) 0 (0)a 0 (0) 0 (0)a 0 (0) 0 (0) 0 (0)a 3 (3) 17 (0)a 4 (1) 15 (5)a 20 (3)a
62 (2)b 8 (8) 15 (5)b 10 (5) 20 (0)b 28 (22) 5 (0) 0 (0)a 8 (3) 0 (0)b 0 (0) 0 (0)b 3 (3)b
50 (7)b 17 (13) 4 (1) 8 (3) 8 (3) 30 (15) 3 (3) 3 (0)b 35 (25) 0 (0)b 2 (2) 0 (0)b 0 (0)b
2 (2)a 0 (0) 0 (0)a 5 (5) 3 (0)a 3 (3) 2 (2) 3 (0)b 2 (2) 0 (0)b 2 (2)b 0 (0)b 3 (3)bb
Lower case letters refer to dierences between layers/horizons (P < 0.05). Signi®cant treatment eects across all horizons (P < 0.05).
associated with the high worm treatment. Cladosporium cladosporioides, T. polysporum and basidiomycete 955 and sterile darks 893, 876 and 877 were positively and O. echinulatum, Verticillium fungicola var fungicola, M. ramanniana var angulispora, M. an zonata, Mucor 866, and sterile dark 898 were negatively associated with ®nal worm biomass. In the Ah horizon, initial treatment and ®nal worm biomass did not explain any of the variation in the fungal species data (data not shown). In the Bm horizon, ®nal worm biomass (P < 0.01), C-to-N ratio (P < 0.01), yr 2 (P < 0.001), organic matter content (P < 0.001) and moisture content (P < 0.001) correlated with the ®rst PCA axis, accounting for 56% of the variation in the fungal species data (data not shown). V. fungicola var fungicola, O. ¯avum, O. an. citrinum, Thysanophora penicilloides, Geomyces an pannorum, Phialophora 938 and Rhinocladiella 845 were negatively associated with ®nal worm biomass.
4. Discussion In terms of gross characteristics the fungal communities of the soil pro®le we studied are similar to those found in a study of Scots pine soil by SoÈderstroÈm and BaÊaÊth (1978). In general, the same genera were abundant, although Trichoderma, and not Mortierella was dominant in our study. The similarity index between adjacent horizons was similar in both studies, although in our study the fungal communities in the Ah and Bm horizons were much less similar than between the Ae and Bm horizons in the Swedish study (SoÈderstroÈm
and BaÊaÊth, 1978). This may re¯ect the fact that at our study site the Ah horizon was in the early stages of development. Our hypothesis that the activities of D. octaedra over 2 yr would homogenize the soil pro®le, reducing spatial heterogeneity and thus species richness and diversity, was supported by the data. After 2 yr of earthworm activities, the organic layers in the high worm treatment were almost completely homogenized, resulting in a thin L1 layer above a layer of casts overlaying the developing Ah horizon. Consistent with our hypothesis, high worm activity accounted for almost all the variation in fungal diversity and richness in the FH layer, and Ah and Bm horizons and high worm numbers or biomass were positively correlated with fungal dominance and negatively correlated with species richness and diversity. The fungal similarity data indicated that the eects on the fungal community were more intense in the high than in the low worm treatment at both times. Evidence that the intense activity of the earthworms in the high worm treatment changed the fungal species composition comes from the species abundance data. At both sampling times, 3 fungal taxa occurred only in the low worm treatment and 8 taxa occurred only in the high worm treatment. In addition, 21 taxa occurred only in the low worm treatment and 37 taxa occurred only in the high worm treatment at one sampling time. Of these, while many only occurred once, and were probably not major components of the fungal community or indicators of community change, the other taxa (3 and 8, respectively), which occurred more frequently suggest that the composition of the fungal community was changing due to the activities of D. octaedra.
M.A. McLean, D. Parkinson / Soil Biology & Biochemistry 32 (2000) 351±360
357
Fig. 1. PCA of fungal species in the FH layer in low and high worm plots 1 and 2 yr following the introduction of worms. Environmental codes as follows: H high worm treatment; WORM WT worm biomass. Fungal species codes are as follows: a824 Paecilomyces farinosus; b955 basidiomycete 955 and sterile darks 877 and 893 and sterile hyaline 940; c852 Cladosporium cladosporioides; d876 sterile dark 876; d877 sterile dark 877 see b955; d880 sterile dark 880; d881 sterile dark 881; d893 sterile dark 893 see b955; d898 sterile dark 898; d943 sterile dark 943; h926 sterile hyaline 926; h940 sterile hyaline 940 see b955; h944 sterile hyaline 944; h958 sterile hyaline 958; k902 black yeast 902; m856 Mortierella ramanniana var ramanniana; m857 M. ramanniana var angulispora see o823; m860 Mortierella 860 and Mucor an. plumbeus; m861 M. parvispora; m862 M. humilis; m864 Mortierella 864; o823 Oidiodendron echinulatum and M. ramanniana var angulispora and Mortierella an zonata and Mucor 866; o830 O. griseum; o836 O. tenuissimum; p806 Penicillium montanense; p807 P. lanosum; p812 P. janczewskii; p813 P. janthinellum; p814 P. citreonigrum; s842 sterile yellow 842; s871 Tolypocladium niveum; s888 Volutella 888; t601 Trichoderma 601; t802 T. koningii; t803 T. polysporum; t911 T. longibrachiatum; t912 T. an. fertile; v828 Verticillium fungicola var fungicola; z866 Mucor 866 and Mortierella an zonata see o823; z910 Mucor an. plumbeus see m860.
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Potential mechanisms by which D. octaedra could aect fungal (microbial) species composition are: 1. comminution; 2. casting, and 3. grazing. 1. Comminution of litter by earthworms increases the surface area of the material, allowing increased bacterial access to the material and increasing the bacterial-to-fungal ratio (Scheu and Parkinson, 1994a). As well as favouring the development of bacteria, comminution by earthworms may disrupt fungal hyphal networks, reduce the growth and activity of many fungi and favour fast growing species (Visser, 1985). High levels of worm activity in the FH layer and Ah and Bm horizons were positively correlated with increased dominance by Trichoderma polysporum, re¯ecting the tolerance of this fast-growing species to the disruptive activities of D. octaedra. Other fast-growing species associated with high worm numbers or biomass were T. koningii, basidiomycete 955 and sterile darks 876 and 877. In addition, the data show that 29% of those species occurring only in the low worm treatment were fast growing and 47% of those species occurring only in the high worm treatment were fast growing. Although these growth rates were on agar and the correlation between growth on agar and on natural substrates is subject to argument, they suggest that this may be a partial explanation of the observed eects of worm activities. In the FH layer, decreases in the frequency of occurrence of both subspecies of M. ramanniana with increasing worm biomass and the negative association of several Zygomycetes (Mucor an. plumbeus, Mu. an. zonata, Mucor 866, Mortierella 860 and 864 and M. ramanniana var angulispora ) with worm biomass or numbers may re¯ect their inability to tolerate worm activities. Zygomycetes have few septa and are therefore more susceptible than other groups of fungi to cell content leakage when the hyphae are damaged. Septate fungi can block o damaged parts of the hypha, preventing cytoplasm loss by septum formation or plugging an existing septum (Cooke and Whipps, 1993). Other species which were negatively associated with the ®nal worm biomass (O. echinulatum and sterile dark 898), or whose frequency of occurrence decreased with increasing worm biomass (P. lanosum and O. griseum ), are relatively slow growing, and thus may not have been able to compensate for the disruption due to worm activities. 2. Casting includes physical and chemical changes to material in casts relative to the original materials. In our experiment in the high worm plots the L2, F and H layers were thoroughly mixed and homogenized. The activities of D. octaedra in the F and
H layers reduced the physical heterogeneity from the fragmented and diverse particle types present in the F and H layers to worm casts. McLean and Parkinson (1998) suggested that decreases in the abundance of aggregated fungal taxa in mesocosms were an indication that homogenization of the organic layers by D. octaedra resulted in a reduction of niches for fungal specialists. The observed changes in fungal species composition at our study site may be further evidence of this although fungal species aggregation could not be assessed. In laboratory experiments D. octaedra and another epigeic earthworm, Lumbricus rubellus, increased the leaching of mineral nutrients (N, Na+, K+, Ca2+, PO3ÿ 4 ) and increased the ammonium-to-nitrate ratio in forest ¯oor materials (Anderson et al., 1983; Haimi and Huhta, 1990; Scheu and Parkinson, 1994a). In ®eld experiments where nutrient ¯uxes were not measured D. octaedra decreased the mineral N content of the L/F layers (Scheu and Parkinson, 1994b) and decreased the total N content of the L and FH layers and Ah horizon in the ®rst year (McLean and Parkinson, 1997b). These decreases in nutrient pools may re¯ect increased nutrient leaching due to the activities of the worms. Several studies have shown the ÿ importance of NH+ 4 and NO3 in limiting the distribution of soil fungi and have demonstrated that ÿ fungi respond dierently to NH+ 4 and NO3 (e.g. Widden, 1986). Since some species of the Mucorales are unable to utilize nitrate (Dix and Webster, 1995), changes in the ammonium-to-nitrate ratio in our experiment may have contributed to the negative eects of D. octaedra on several species of this group in the FH layer. Leaching of mineral N from the FH layer and Ah horizon may have contributed to the increases in abundance of T. koningii and M. ramanniana var. angulispora observed in the Bm horizon in 1994. That the distribution of T. koningii has been related to high nitrogen and ammonium concentrations (Park, 1976; Widden, 1986) tends to support this idea. McLean and Parkinson (1997b) suggested that decreases in organic matter content and total N and C resulted in increased respiration and metabolic quotient (qCO2) over time, re¯ecting adjustments by the microbial community to decreases in C availability in the casts of D. octaedra. While stabilization of soil carbon may be promoted by clay-associated carbohydrates which are more abundant in endogeic earthworm casts than in the surrounding soil (Shaw and Pawluk, 1986a,b) it is unlikely that epigeic earthworms would have a similar eect on C availability. Endogeic earthworms ingest highly decomposed amorphous organic matter associated with mineral material (Edwards and
M.A. McLean, D. Parkinson / Soil Biology & Biochemistry 32 (2000) 351±360
Bohlen, 1996). D. octaedra, like other epigeic earthworms, feeds on relatively undecomposed litter (Edwards and Bohlen, 1996) and epigeic gut passage results in comminuted but not chemically transformed organic materials (Ponge, 1991; Ziegler and Zech, 1992). In the pine forest in our study, it is clear that this species does mix organic and mineral material but this is not likely to result in the binding of carbon by clays as is found in endogeic earthworm casts. Changes in microbial activity observed by McLean and Parkinson (1997b) therefore may re¯ect stimulation of microbial growth as the microbial biomass is consumed by the earthworms or changes in fungal species composition. Earthworm activities can represent a major perturbation to litter and soil fungi and most fungal hyphal biomass is destroyed during gut passage through anecic and endogeic earthworms (e.g. Edwards and Bohlen, 1996) thus creating new substrates available for fungal colonization. Passage through endogeic and anecic earthworm guts signi®cantly alters fungal spore viability, decreasing the viability of many fungal species while increasing the viability of other fungal species (e.g. Edwards and Bohlen, 1996; Moody et al., 1996). If passage through epigeic earthworm guts has similar eects on hyphal and spore viability, dierential survival of spores able to colonize newly available substrates in earthworm casts may be an important mechanism in¯uencing the fungal community. Another consequence of the killing of fungal hyphal biomass during gut passage may be casts enriched in chitin, which is an important component in fungal cell walls (Cooke and Rayner, 1984). This might provide an ideal substrate for chitinolytic fungi such as species of the genera Trichoderma, Penicillium and Mortierella (Cooke and Rayner, 1984; Domsch et al., 1993) and may be a partial explanation for the observed positive association of T. koningii, T. polysporum and P. montanense with high worm activities in the FH layer. 3. Visser (1985); Brown (1995); Moody et al. (1995) and others have postulated that selective grazing is an important mechanism by which earthworms aect fungal communities. Although earthworms can select substrates inoculated with dierent species of fungi (e.g. Cooke, 1983; Moody et al., 1996), the large scale eects of this have not yet been demonstrated. The activities of D. octaedra at high densities and biomasses in the ®eld aected the fungal community by: (i) increasing fungal dominance, decreasing diversity and species richness in the FH layer and Ah and Bm horizons; (ii) dierentiat-
359
ing the fungal communities in the FH layer from those in the L layer and Bm horizons; (iii) increasing the similarity between the fungal communities in the FH layer and the Ah horizon; (iv) changing the fungal species composition.
Acknowledgements This work was supported by an NSERC Operating Grant to D.P. and by the Biodiversity Grants Program, through the joint eorts of the sportsmen of Alberta and the Alberta Department of Environmental Protection, Fish and Wildlife Trust Fund. Our thanks to D. Kolodka for technical assistance.
References Anderson, J.M., Ineson, P., Huish, S.A., 1983. The eects of animal feeding activities on element release from deciduous forest litter and soil organic matter. In: Lebrun, Ph., AndreÂ, H.M., De Medts, A., GreÂgoire-Wibo, C., Wauthy, G. (Eds.), New Trends in Soil Biology. Dieu-Brichast, Louvaine-la-Neuve, pp. 87±100. Brown, G.G., 1995. How do earthworms aect micro¯oral and faunal community diversity? Plant and Soil 170, 209±231. Cooke, A., 1983. The eects of fungi on food selection by Lumbricus terrestris L. In: Satchell, J.E. (Ed.), Earthworm Ecology: From Darwin to Vermiculture. Chapman and Hall, New York, pp. 365±373. Cooke, R.C., Rayner, A.D.M., 1984. Ecology of Saprotrophic Fungi. Longman, London. Cooke, R.C., Whipps, J.M., 1993. Ecophysiology of Fungi. Blackwell Scienti®c Publications, Oxford. Dix, N.J., Webster, J., 1995. Fungal Ecology. Chapman and Hall, London. Domsch, K.-H., Gams, W., Anderson, T.-H., 1993. Compendium of Soil Fungi. IHW-Verlag, Eching (reprint of 1980 edition+supplement). Doube, B.M., Brown, G.G., 1998. Life in a complex community: functional interactions between earthworms, organic matter, microorganisms and plants. In: Edwards, C.A. (Ed.), Earthworm Ecology. Soil and Water Conservation Society, Ankeny, pp. 179± 211. Edwards, C.A., Bohlen, P.J., 1996. Biology and Ecology of Earthworms, 3rd ed. Chapman and Hall, London. Haimi, J., Huhta, V., 1990. Eects of earthworms on decomposition processes in raw forest humus soil: a microcosm study. Biology and Fertility of Soils 10, 178±183. Kempson, D., Lloyd, M., Ghelardi, R., 1963. A new extractor for woodland litter. Pedobiologia 3, 1±21. Magurran, A.E., 1988. Ecological Diversity and Its Measurement. Princeton University Press, Princeton. McLean, M.A., Parkinson, D., 1997a. Changes in structure, organic matter and microbial activity in pine forest soil following the introduction of Dendrobaena octaedra (Oligochaeta, Lumbricidae). Soil Biology & Biochemistry 29, 537±539. McLean, M.A., Parkinson, D., 1997b. Soil impacts of the epigeic earthworm Dendrobaena octaedra on organic matter and microbial activity in lodgepole pine forest. Canadian Journal of Forest Research 27, 1907±1913.
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McLean, M.A., Parkinson, D., 1998. Impacts of the epigeic earthworm Dendrobaena octaedra on microfungal community structure in pine forest ¯oor: a mesocosm study. Applied Soil Ecology 8, 61±75. McLean, M.A., Kolodka, D.U., Parkinson, D., 1996. Survival and growth of Dendrobaena octaedra (Savigny) in pine forest ¯oor materials. Pedobiologia 40, 281±288. Moody, S.A., Briones, M.J.I., Piearce, T.G., Dighton, J., 1995. Selective consumption of decomposing wheat straw by earthworms. Soil Biology & Biochemistry 27, 1209±1213. Moody, S.A., Piearce, T.G., Dighton, J., 1996. Fate of some fungal spores associated with wheat straw decomposition on passage through the guts of Lumbricus terrestris and Aporrectodea longa. Soil Biology & Biochemistry 28, 533±537. Niklas, J., Kennel, W., 1981. The role of the earthworm, Lumbricus terrestris (L.) in removing sources of phytopathogenic fungi in orchards. Gartenbauwissenschaft 46, 138±142. Park, D., 1976. Carbon and nitrogen levels as factors in¯uencing fungal decomposers. In: Anderson, J.M., Macfadyen, J. (Eds.), The Role of Terrestrial and Aquatic Organisms in Decomposition Processes. Blackwell, Oxford, pp. 41±59. Parkinson, D., 1994. Filamentous fungi. In: Weaver, R.W., Angle, J.S., Bottomley, P.S. (Eds.), Methods of Soil Analysis. 2. Microbiological and Biochemical Properties. Soil Science Society of America, Madison, pp. 329±350. Parkinson, D., McLean, M.A., 1998. Impacts of earthworms on the community structure of other biota in forest soils. In: Edwards, C.A. (Ed.), Earthworm Ecology. Soil and Water Conservation Society, Ankeny, pp. 213±226. Ponge, J.F., 1991. Successsion of fungi and fauna during decomposition of needles in a small area of Scots pine litter. Plant and Soil 138, 99±113.
Scheu, S., Parkinson, D., 1994a. Eects of earthworms on nutrient dynamics, carbon turnover and microorganisms in soils from cool temperate forests of the Canadian Rocky Mountains: laboratory studies. Applied Soil Ecology 1, 113±125. Scheu, S., Parkinson, D., 1994b. Eects of invasion of an aspen forest (Canada) by Dendrobaena octaedra (Lumbricidae) on plant growth. Ecology 75, 2348±2361. Shaw, C., Pawluk, S., 1986a. The development of soil structure by Octolasion tyrtaeum, Aporrectodea turgida and Lumbricus terrestris in parent materials belonging to dierent textural classes. Pedobiologia 29, 327±339. Shaw, C., Pawluk, S., 1986b. Faecal microbiology of Octolasion tyrtaeum, Aporrectodea turgida and Lumbricus terrestris and its relation to the carbon budgets of three arti®cial soils. Pedobiologia 29, 377±389. SoÈderstroÈm, B.E., BaÊaÊth, E., 1978. Soil microfungi in three Swedish coniferous forests. Holarctic Ecology 1, 62±72. Visser, S., 1985. The role of soil invertebrates in determining the composition of soil microbial communities. In: Fitter, A.H., Atkinson, D., Read, D.J., Usher, M.B. (Eds.), Ecological Interactions in Soil. Blackwell Scienti®c Publications, Oxford, pp. 287±317. Widden, P., 1986. Functional relationships between Quebec forest soil microfungi and their environment. Canadian Journal of Botany 64, 1424±1432. Worrall, J.J., 1991. Media for selective isolation of hymenomycetes. Mycologia 83, 296±302. Ziegler, F., Zech, W., 1992. Chemical changes of beech litter during passage through the gut of the lumbricid earthworm Eisenia fetida (Sav.). Zeitschrift fuer P¯anzenernaÈhrung und Bodenkunde 155, 69±70.
www.elsevier.com/locate/soilbio
Field evidence of the eects of the epigeic earthworm Dendrobaena octaedra on the microfungal community in pine forest ¯oor M.A. McLean a,*, D. Parkinson b a
Louis Calder Center, Fordham University, 53 Whippoorwill Rd., Armonk, NY 10504, USA Department of Biological Sciences, University of Calgary, Calgary, Alta., Canada T2N 1N4
b
Accepted 6 September 1999
Abstract The eects of the invasion of the epigeic earthworm Dendrobaena octaedra on the forest ¯oor microfungal community were studied in a 90 yr old lodgepole pine forest over 2 yr. Fungi were isolated from the L and FH layers and the Ah and Bm horizons 1 and 2 yr after the introduction of earthworms to plots. High density and biomass of D. octaedra correlated positively with fungal dominance and negatively with fungal richness and diversity in the FH layer and the Ah and Bm horizons. High worm density and biomass dierentiated the fungal communities in the FH layer from those in the L layer and Bm horizons and increased the similarity between the fungal communities in the FH layer and the Ah horizon. Earthworm activities appeared to favour the presence of faster growing fungal taxa. # 2000 Elsevier Science Ltd. All rights reserved. Keywords: Fungal community; Earthworm; Dendrobaena octaedra; Soil fungi; Litter fungi
1. Introduction It is well known that earthworms, through their channelling and mixing of organic matter and mineral soil and comminution of organic matter, have signi®cant eects on soil structure and soil chemical properties and thus on microbial activity and on microbial populations (Brown, 1995; Edwards and Bohlen, 1996; Doube and Brown, 1998). Much less is known of the eects of earthworms on microbial communities (Parkinson and McLean, 1998), although we do know that earthworm casts are a favourable environment for fungal growth (e.g. Brown, 1995; Edwards and Bohlen, 1996; Doube and Brown, 1998), burial of leaf litter by anecic earthworms can signi®cantly reduce phytopathogenic fungal propagules (Niklas and Kennel,
* Corresponding author. Tel.: +1-914-273-3078 x18; fax: +1-914273-2167. E-mail address: [email protected] (M.A. McLean).
1981) and that earthworms can graze selectively on fungi (e.g. Cooke, 1983; Moody et al., 1995). A recent invasion of the epigeic earthworm, Dendrobaena octaedra (Savigny) into lodgepole pine forest ¯oors in southwest Alberta, Canada, has provided the opportunity to investigate the eects of an epigeic earthworm on microbial activity and the fungal community. Two approaches were used in our investigation: short-term (6 months) laboratory studies (McLean and Parkinson, 1997a, 1998) and longer-term (2 yr) ®eld studies (the present experiment; McLean and Parkinson, 1997b). In this soil the activities of D. octaedra have signi®cantly altered the organic layers and upper mineral horizons (McLean and Parkinson, 1997a). These physical changes were accompanied by decreased microbial biomass and fungal-to-bacterial ratio in laboratory studies (Scheu and Parkinson, 1994a; McLean and Parkinson, 1997a), and decreased basal respiration and metabolic quotient in the ®eld (McLean and Parkinson, 1997b). In laboratory mesocosms, the activities of D. octaedra increased fungal
0038-0717/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 8 - 0 7 1 7 ( 9 9 ) 0 0 1 6 1 - 3
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M.A. McLean, D. Parkinson / Soil Biology & Biochemistry 32 (2000) 351±360
species richness, diversity and number of isolates per particle in the short term (6 months) (McLean and Parkinson, 1998). This was attributed to increased spatial heterogeneity, through the addition of casts and other products of earthworm activities to the types of organic substrates already present in the soil. The organic layers in mesocosms containing high numbers of earthworms were completely homogenized (i.e. the organic layers were composed solely of casts). We hypothesize that the longer term (2 yr) eects of these worms will be reduced spatial heterogeneity and therefore decreased fungal species richness and diversity. Knowing that D. octaedra attains maximum growth in the FH layer and Ah horizon (McLean et al., 1996), and that the physical eects of its mixing activities are strongest in these horizons, we hypothesize that its eects on the fungal community will be most intense in these horizons. We have investigated the eects of D. octaedra on the fungal community in the ®eld over 2 yr, in the context of the previous hypotheses.
2. Materials and methods 2.1. Site description This experiment was conducted in a 90 yr old lodgepole pine (Pinus contorta Loud. var. latifolia Engelm.) forest ¯oor in the Kananaskis Valley in the Rocky Mountains of southwestern Alberta, Canada. For a more detailed description see McLean and Parkinson (1997b). 2.2. Experimental design Five pairs of plots 1 by 2 m were set up in August 1993 in a part of the pine forest which surveys had shown to be free of earthworms. Within each pair of plots, two treatments (worms added, no worms added) were randomly assigned. To each of the worm-treated plots 250 immature and 70 mature specimens mÿ2 of Dendrobaena octaedra were added, with a total biomass of 3.3 g d.w. mÿ2. Plots were sampled in September 1994 and September 1995 for assessment of worm abundance and biomass and the occurrence of fungal taxa. The F and H layers were not separated in this experiment since in all cases worm activities had mixed them together. An Ah horizon had developed in one of the plots at 1 yr and in both plots at 2 yr in the high worm treatment and had not formed in the low worm treatment plots at either time.
2.3. Earthworm abundance and biomass Earthworms were heat extracted (Kempson et al., 1963) from 10.5 cm dia cores taken from each plot at each sampling time, and classi®ed as small immatures (<1 cm length), large immatures, matures (clitellate) and aclitellate adults. Oven dry weights of each size class were used to estimate earthworm biomass. Mean biomass of a mature worm was 27 mg d.w. Worm biomass (abundance) in the high worm plots were 13.6 (462) and 27.7 g d.w. (1385) mÿ2 in 1994 and 39.9 (3233) and 19.6 g d.w. (3349) mÿ2 in 1995. No worms were recorded in the low worm plots in September 1994 and September 1995, although a few worms were observed in these plots during the summer. 2.4. Fungi Three subsamples from each replicate of each plot were collected at both sampling times for assessment of the fungal community. These subsamples were separated into layers/horizons (L, FH, Ah (present only in the high worm treatment) and Bm) and then bulked to form composite samples of each layer/horizon from which fungi were isolated using a washing procedure (Parkinson, 1994). Fifty particles (<0.2 mm) were plated, one per plate, from each replicate layer/horizon. Of these, 30 were plated on 2% malt extract agar plus antibiotics (0.1% streptomycin and 0.05% aureomycin) and 20 were plated on malt extract agar plus antibiotics with 5 mg benomyl lÿ1 to permit the isolation of basidiomycetes (Worrall, 1991). Since the potential number of plates and of fungal isolates would have been very large and dicult to handle if all the plots had been sampled, only the fungal communities in the two plots with the highest and lowest worm biomasses at each sampling time were investigated. Although surveys had shown the area to be free of worms, worms were already invading the forest during the experiment. The September sampling in both years did not reveal the presence of any worms in the low worm biomass plots but a few worms and casts were observed in these plots in both years. Therefore the two treatments will be referred to as the high worm and low worm treatments. 2.5. Statistical analysis Fungal community characteristics (similarity, number of fungal isolates per particle, species richness, dominance (d ) and diversity (1/D )) were calculated from the percent frequency of occurrence data (Magurran, 1988). Number of fungal isolates per particle was the mean number of taxa isolated per particle in each replicate of each treatment. Data were analysed in two ways: using ANCOVA
M.A. McLean, D. Parkinson / Soil Biology & Biochemistry 32 (2000) 351±360 Table 1 Morisita±Horn quantitative similarity index for the fungal communities 1 and 2 yr after the introduction of worms to the ®eld plots 1 yr
2 yr
Low vs. high worms L FH Bm
0.751 0.817 0.754
0.612 0.872 0.486
Low worms L vs. FH FH vs. Bm
0.439 0.540
0.573 0.383
High worms L vs. FH FH vs. Ah FH vs. Bm Ah vs. Bm
0.196 0.495 0.492 0.528
0.099 0.854 0.240 0.312
with ®nal worm biomass as the covariate to take into account both the initial treatment applied and the dierences in ®nal worm biomass; and using principal components analysis (PCA) followed by correlation of the extracted axes with environmental variables (initial treatment, ®nal worm biomass, organic matter content, moisture content, pH, C-to-N ratio). Since the environmental constraints and worm activities were dierent in the dierent layers and horizons sampled, the PCA analysis was conducted on each layer or horizon separately. 3. Results 3.1. Eects on fungal community structure A total of 143 taxa were isolated in the whole study. The fungal communities in the L layer and the Bm horizon in both worm treatments were less similar in yr 2 while those in the FH were more similar (Table 1). The fungal communities in the L and FH layers in the low worm treatment were more similar in yr 2 and those in the FH and Bm horizon were less similar (Table 1). In the high worm treatment, the fungal communities in the L and FH layers were very dierent at both sampling times. In the high worm treatment the fungal communities in the FH and the Ah were more similar in yr 2 while those in the FH and the Ah were less similar to that in the Bm horizon. In the low worm treatment the fungal communities in the L and FH layers and the FH layer and Bm horizon were more similar than those in the same layers at high worm biomass. None of the community characteristics (number of fungal isolates per particle, richness, dominance (d ), diversity (1/D )) calculated were aected by treatment or ®nal worm biomass in either year (Table 2). In the L layer neither ®nal worm biomass or initial
353
treatment aected the fungal community properties. The C-to-N ratio ( p < 0.05) correlated with the ®rst PCA axis which accounted for 99% of the variation in the fungal community data (data not shown). In this layer the C-to-N ratio correlated positively with fungal dominance and negatively with diversity and richness. In the FH layer, the high worm treatment ( p < 0.05) and C-to-N ratio ( p < 0.05) correlated with the ®rst PCA axis which accounted for 98% of the variation in the fungal community characteristics (data not shown). The high worm treatment positively correlated with fungal dominance and negatively with diversity and richness, while the C-to-N ratio correlated positively with diversity and richness and negatively with dominance. In the Ah and Bm horizons, ®nal worm biomass correlated ( p < 0.05) with the ®rst PCA axis, accounting for 97 and 99% of the variation in fungal community data, respectively (data not shown). In both horizons, ®nal worm biomass was positively correlated with fungal dominance and negatively correlated with fungal diversity. In the Ah horizon, ®nal worm biomass was also negatively correlated with fungal species richness. 3.2. Eects on fungal species abundance At 1 yr Trichoderma koningii, Penicillium lanosum, Oidiodendron griseum and basidiomycete 955 occurred more frequently in all layers/horizons in the high worm treatment than in the low worm treatment (Table 3). Percent frequency of occurrence of P. lanosum, O. griseum, Mortierella ramanniana var. ramanniana and M. ramanniana var. angulispora declined in the FH layer with increasing ®nal worm biomass. Percent frequency of occurrence of T. koningii and M. ramanniana var. angulispora increased in the Bm horizon with increasing ®nal worm biomass. Percent frequency of occurrence of basidiomycete 955 increased in the FH layer and decreased in the L layer with increasing ®nal worm biomass. At 2 yr sterile dark 880 occurred more frequently and sterile yellow 842 occurred less frequently in the low than in the high worm treatment (Table 4). None of the taxa were aected by ®nal worm biomass. In the L layer, none of the environmental variables, including initial treatment and ®nal worm biomass, correlated signi®cantly with the PCA axes (data not shown). In the FH layer, the high worm treatment (P < 0.05) and ®nal worm biomass (P < 0.05) correlated with the ®rst PCA axis, accounting for 37% of the variation in the fungal species data (Fig. 1). Trichoderma koningii, Penicillium montanense, basidiomycete 955 and sterile darks 876, 877 and 893 were positively and Mucor an. plumbeus, Mortierella 860 and 864, sterile hyaline 926 and sterile dark 880 were negatively
2.3 (0.4) 1.7 (0.0)
33 (1) 25 (2)
7.4 (0.3) 6.8 (0.1)
0.11 (0.03) 0.15 (0.01)
30.6 (10.6) 18.8 (0.7)
Stot Low High
S8 Low High
d Low High
1/D Low High 14.4 (4.4) 11.5 (0.9)
0.20 (0.03) 0.22 (0.04)
6.6 (0.4) 6.5 (0.0)
32 (5) 28 (2)
3.1 (0.2) 3.0 (0.2)
ND 11.6 (ÿ)
ND 0.24 (ÿ)
ND 6.3 (ÿ)
ND 33 (ÿ)
ND 3.5 (ÿ)
25.1 (10.5) 20.9 (5.7)
0.13 (0.03) 0.15 (0.05)
7.0 (0.6) 6.9 (0.3)
31 (5) 25 (8)
2.6 (0.5) 2.2 (1.1)
Bm
21.9 (0.0) 29.2 (8.0)
0.16 (0.02) 0.05 (0.04)
7.5 (0.1) 7.3 (0.2)
25 (2) 30 (2)
1.8 (0.1) 1.8 (0.1)
L
Ah
L
FH
2 yr
1 yr
Isolates Low High
Treatment
11.6 (2.0) 9.4 (1.4)
0.20 (0.04) 0.27 (0.05)
6.2 (0.3) 6.1 (0.1)
22 (2) 22 (4)
2.8 (0.3) 2.4 (0.4)
FH
ND 10.8 (2.0)
ND 0.23 (0.04)
ND 6.3 (0.3)
ND 28 (5)
ND 2.6 (0.4)
Ah
20.7 (0.9) 31.1 (ÿ)
0.15 (0.01) 0.12 (0.01)
6.9 (0.0) 7.8 (0.2)
14 (2) 18 (10)
0.8 (0.2) 0.9 (0.6)
Bm
Table 2 Mean (standard error) number of fungal isolates per particle plated, fungal species richness (Stot: total number of species, S8: number of species based on sample size of 8 from rarefaction), fungal dominance (d ) and fungal diversity (1/D ) in the L and FH layers and the Ah and Bm horizons 1 and 2 yr after the introduction of Dendrobaena octaedra into ®eld plots n 2). ND: not determined; the Ah horizon was not present in the low worm treatment. Neither initial treatment nor ®nal worm biomass aected the fungal community parameters (P > 0.95)
354 M.A. McLean, D. Parkinson / Soil Biology & Biochemistry 32 (2000) 351±360
b
a
Lower case letters refer to dierences between layers/horizons (P < 0.05). Signi®cant treatment eects across all horizons (P < 0.05).
Trichoderma koningii Oudem. T. polysporum (Link: Fr.) Rifai Penicillium montanense Christensen and Backus P. lanosum Westling Mortierella ramanniana (MoÈller) Linnem. var. ramanniana M. ramanniana var. angulispora (Naumov) Linnem. M. vinacea Dixon-Stewart M. parvispora Linnem. M. humilis Linnem. Oidiodendron griseum Robak Geomyces pannorum (Link) Sigler and Carmichael var. vinaceus (Dal Vesco) van Oorshot Tolypocladium niveum (Rostrup) Bissett Sterile dark 875 Sterile dark 880 Sterile dark 889 Basidiomycete 955
Taxon
2 (2) 20 (17)a 0 (0) 0 (0) 2 (2)a 0 (0) 0 (0) 5 (5) 5 (0) 0 (0) 0 (0) 3 (3) 18 (2)a 7 (0) 11 (4)a 6 (1)a
2 (2) 63 (13)b 2 (2) 0 (0) 35 (0)b 3 (3) 3 (3) 23 (8) 35 (15) 3 (0) 3 (0) 13 (3) 2 (2)b 8 (2) 0 (0)b 0 (0)b
ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND
0 (0) 23 (3)a 27 (17) 2 (2) 7 (3)a 0 (0) 28 (22) 13 (7) 9 (6) 3 (3) 3 (0) 8 (8) 0 (0)b 3 (0) 0 (0)b 0 (0)
Bm
0 (0) 7 (3)a 0 (0) 0 (0)a 0 (0)a 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)a 2 (2) 0 (0) 20 (0)a 0 (0) 10 (7)a 10 (10)a ÿ
L
Ah
L
FH
High worms
Low worms
Ah 27 40 20 3 17 3 5 7 25 3 13 85 0 0 0 0
FH 7 (0)aa 63 (10)b 13 (0) 18 (5)b ÿ 40 (15)b ÿ 7 (7) ÿ 15 (0) 8 (8) 8 (3) 22 (15)b ÿ 2 (2) 3 (3) 3 (3)b 2 (2)a 0 (0)b 5 (5)b +
(ÿ) (ÿ) (ÿ) (ÿ) (ÿ) (ÿ) (ÿ) (ÿ) (ÿ) (ÿ) (ÿ) (ÿ) (ÿ) (ÿ) (ÿ) (ÿ)
17 (17)b +b 10 (10)a 10 (10) 2 (2)ab 5 (5)a 10 (10) + 18 (8) 10 (0) 10 (0) 2 (2)ab 5 (5) 8 (8) 0 (0)b 10 (3)b 0 (0)b 0 (0)b
Bm
Table 3 Mean (standard error) percent frequency of occurrence of most abundant fungal taxa in the L and FH layers and the Ah and Bm horizons 1 yr after the introduction of worms to the ®eld plots n 2). ND: not determined; the Ah horizon was not present in the low worm treatment; only one of the plots in the high worm treatment developed an Ah horizon. +, ÿ refer to positive and negative eects of increasing ®nal worm biomass within horizons across treatments (P = 0.05)
M.A. McLean, D. Parkinson / Soil Biology & Biochemistry 32 (2000) 351±360 355
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M.A. McLean, D. Parkinson / Soil Biology & Biochemistry 32 (2000) 351±360
Table 4 Mean (standard error) percent frequency of occurrence of most abundant fungal taxa in the L and FH layers and the Ah and Bm horizons 2 yr after the introduction of worms to the ®eld plots n 2). ND: not determined; the Ah horizon was not present in the low worm treatment Taxon
Trichoderma polysporum Mortierella ramanniana var. ramanniana M. ramanniana var. angulispora M. vinacea M. parvispora M. humilis Verticillium fungicola var. fungicola Oidiodendron griseum Tolypocladium niveum Sterile dark 875 Sterile dark 880 Sterile dark 919 Sterile yellow 842 a b
Low worms
High worms
L
FH
Ah
Bm
L
FH
Ah
Bm
28 (2)aa 0 (0) 3 (3)a 0 (0) 5 (5)a 5 (0) 0 (0)a 0 (0) 4 (1) 12 (2)a 5 (2) 12 (2)a 13 (3)a
58 (18)b 18 (3) 10 (0)b 8 (2) 13 (3)b 43 (8) 28 (8)b 2 (2) 17 (13) 2 (2)b 10 (3) 0 (0)b 0 (0)b
ND ND ND ND ND ND ND ND ND ND ND ND ND
0 (0)a 5 (5) 3 (3)a 5 (5) 5 (5)a 8 (3) 0 (0)a 0 (0) 0 (0) 0 (0)b 3 (0) 0 (0)b 0 (0)b
3 (3)a 0 (0) 0 (0)a 0 (0) 0 (0)a 0 (0) 0 (0) 0 (0)a 3 (3) 17 (0)a 4 (1) 15 (5)a 20 (3)a
62 (2)b 8 (8) 15 (5)b 10 (5) 20 (0)b 28 (22) 5 (0) 0 (0)a 8 (3) 0 (0)b 0 (0) 0 (0)b 3 (3)b
50 (7)b 17 (13) 4 (1) 8 (3) 8 (3) 30 (15) 3 (3) 3 (0)b 35 (25) 0 (0)b 2 (2) 0 (0)b 0 (0)b
2 (2)a 0 (0) 0 (0)a 5 (5) 3 (0)a 3 (3) 2 (2) 3 (0)b 2 (2) 0 (0)b 2 (2)b 0 (0)b 3 (3)bb
Lower case letters refer to dierences between layers/horizons (P < 0.05). Signi®cant treatment eects across all horizons (P < 0.05).
associated with the high worm treatment. Cladosporium cladosporioides, T. polysporum and basidiomycete 955 and sterile darks 893, 876 and 877 were positively and O. echinulatum, Verticillium fungicola var fungicola, M. ramanniana var angulispora, M. an zonata, Mucor 866, and sterile dark 898 were negatively associated with ®nal worm biomass. In the Ah horizon, initial treatment and ®nal worm biomass did not explain any of the variation in the fungal species data (data not shown). In the Bm horizon, ®nal worm biomass (P < 0.01), C-to-N ratio (P < 0.01), yr 2 (P < 0.001), organic matter content (P < 0.001) and moisture content (P < 0.001) correlated with the ®rst PCA axis, accounting for 56% of the variation in the fungal species data (data not shown). V. fungicola var fungicola, O. ¯avum, O. an. citrinum, Thysanophora penicilloides, Geomyces an pannorum, Phialophora 938 and Rhinocladiella 845 were negatively associated with ®nal worm biomass.
4. Discussion In terms of gross characteristics the fungal communities of the soil pro®le we studied are similar to those found in a study of Scots pine soil by SoÈderstroÈm and BaÊaÊth (1978). In general, the same genera were abundant, although Trichoderma, and not Mortierella was dominant in our study. The similarity index between adjacent horizons was similar in both studies, although in our study the fungal communities in the Ah and Bm horizons were much less similar than between the Ae and Bm horizons in the Swedish study (SoÈderstroÈm
and BaÊaÊth, 1978). This may re¯ect the fact that at our study site the Ah horizon was in the early stages of development. Our hypothesis that the activities of D. octaedra over 2 yr would homogenize the soil pro®le, reducing spatial heterogeneity and thus species richness and diversity, was supported by the data. After 2 yr of earthworm activities, the organic layers in the high worm treatment were almost completely homogenized, resulting in a thin L1 layer above a layer of casts overlaying the developing Ah horizon. Consistent with our hypothesis, high worm activity accounted for almost all the variation in fungal diversity and richness in the FH layer, and Ah and Bm horizons and high worm numbers or biomass were positively correlated with fungal dominance and negatively correlated with species richness and diversity. The fungal similarity data indicated that the eects on the fungal community were more intense in the high than in the low worm treatment at both times. Evidence that the intense activity of the earthworms in the high worm treatment changed the fungal species composition comes from the species abundance data. At both sampling times, 3 fungal taxa occurred only in the low worm treatment and 8 taxa occurred only in the high worm treatment. In addition, 21 taxa occurred only in the low worm treatment and 37 taxa occurred only in the high worm treatment at one sampling time. Of these, while many only occurred once, and were probably not major components of the fungal community or indicators of community change, the other taxa (3 and 8, respectively), which occurred more frequently suggest that the composition of the fungal community was changing due to the activities of D. octaedra.
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357
Fig. 1. PCA of fungal species in the FH layer in low and high worm plots 1 and 2 yr following the introduction of worms. Environmental codes as follows: H high worm treatment; WORM WT worm biomass. Fungal species codes are as follows: a824 Paecilomyces farinosus; b955 basidiomycete 955 and sterile darks 877 and 893 and sterile hyaline 940; c852 Cladosporium cladosporioides; d876 sterile dark 876; d877 sterile dark 877 see b955; d880 sterile dark 880; d881 sterile dark 881; d893 sterile dark 893 see b955; d898 sterile dark 898; d943 sterile dark 943; h926 sterile hyaline 926; h940 sterile hyaline 940 see b955; h944 sterile hyaline 944; h958 sterile hyaline 958; k902 black yeast 902; m856 Mortierella ramanniana var ramanniana; m857 M. ramanniana var angulispora see o823; m860 Mortierella 860 and Mucor an. plumbeus; m861 M. parvispora; m862 M. humilis; m864 Mortierella 864; o823 Oidiodendron echinulatum and M. ramanniana var angulispora and Mortierella an zonata and Mucor 866; o830 O. griseum; o836 O. tenuissimum; p806 Penicillium montanense; p807 P. lanosum; p812 P. janczewskii; p813 P. janthinellum; p814 P. citreonigrum; s842 sterile yellow 842; s871 Tolypocladium niveum; s888 Volutella 888; t601 Trichoderma 601; t802 T. koningii; t803 T. polysporum; t911 T. longibrachiatum; t912 T. an. fertile; v828 Verticillium fungicola var fungicola; z866 Mucor 866 and Mortierella an zonata see o823; z910 Mucor an. plumbeus see m860.
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Potential mechanisms by which D. octaedra could aect fungal (microbial) species composition are: 1. comminution; 2. casting, and 3. grazing. 1. Comminution of litter by earthworms increases the surface area of the material, allowing increased bacterial access to the material and increasing the bacterial-to-fungal ratio (Scheu and Parkinson, 1994a). As well as favouring the development of bacteria, comminution by earthworms may disrupt fungal hyphal networks, reduce the growth and activity of many fungi and favour fast growing species (Visser, 1985). High levels of worm activity in the FH layer and Ah and Bm horizons were positively correlated with increased dominance by Trichoderma polysporum, re¯ecting the tolerance of this fast-growing species to the disruptive activities of D. octaedra. Other fast-growing species associated with high worm numbers or biomass were T. koningii, basidiomycete 955 and sterile darks 876 and 877. In addition, the data show that 29% of those species occurring only in the low worm treatment were fast growing and 47% of those species occurring only in the high worm treatment were fast growing. Although these growth rates were on agar and the correlation between growth on agar and on natural substrates is subject to argument, they suggest that this may be a partial explanation of the observed eects of worm activities. In the FH layer, decreases in the frequency of occurrence of both subspecies of M. ramanniana with increasing worm biomass and the negative association of several Zygomycetes (Mucor an. plumbeus, Mu. an. zonata, Mucor 866, Mortierella 860 and 864 and M. ramanniana var angulispora ) with worm biomass or numbers may re¯ect their inability to tolerate worm activities. Zygomycetes have few septa and are therefore more susceptible than other groups of fungi to cell content leakage when the hyphae are damaged. Septate fungi can block o damaged parts of the hypha, preventing cytoplasm loss by septum formation or plugging an existing septum (Cooke and Whipps, 1993). Other species which were negatively associated with the ®nal worm biomass (O. echinulatum and sterile dark 898), or whose frequency of occurrence decreased with increasing worm biomass (P. lanosum and O. griseum ), are relatively slow growing, and thus may not have been able to compensate for the disruption due to worm activities. 2. Casting includes physical and chemical changes to material in casts relative to the original materials. In our experiment in the high worm plots the L2, F and H layers were thoroughly mixed and homogenized. The activities of D. octaedra in the F and
H layers reduced the physical heterogeneity from the fragmented and diverse particle types present in the F and H layers to worm casts. McLean and Parkinson (1998) suggested that decreases in the abundance of aggregated fungal taxa in mesocosms were an indication that homogenization of the organic layers by D. octaedra resulted in a reduction of niches for fungal specialists. The observed changes in fungal species composition at our study site may be further evidence of this although fungal species aggregation could not be assessed. In laboratory experiments D. octaedra and another epigeic earthworm, Lumbricus rubellus, increased the leaching of mineral nutrients (N, Na+, K+, Ca2+, PO3ÿ 4 ) and increased the ammonium-to-nitrate ratio in forest ¯oor materials (Anderson et al., 1983; Haimi and Huhta, 1990; Scheu and Parkinson, 1994a). In ®eld experiments where nutrient ¯uxes were not measured D. octaedra decreased the mineral N content of the L/F layers (Scheu and Parkinson, 1994b) and decreased the total N content of the L and FH layers and Ah horizon in the ®rst year (McLean and Parkinson, 1997b). These decreases in nutrient pools may re¯ect increased nutrient leaching due to the activities of the worms. Several studies have shown the ÿ importance of NH+ 4 and NO3 in limiting the distribution of soil fungi and have demonstrated that ÿ fungi respond dierently to NH+ 4 and NO3 (e.g. Widden, 1986). Since some species of the Mucorales are unable to utilize nitrate (Dix and Webster, 1995), changes in the ammonium-to-nitrate ratio in our experiment may have contributed to the negative eects of D. octaedra on several species of this group in the FH layer. Leaching of mineral N from the FH layer and Ah horizon may have contributed to the increases in abundance of T. koningii and M. ramanniana var. angulispora observed in the Bm horizon in 1994. That the distribution of T. koningii has been related to high nitrogen and ammonium concentrations (Park, 1976; Widden, 1986) tends to support this idea. McLean and Parkinson (1997b) suggested that decreases in organic matter content and total N and C resulted in increased respiration and metabolic quotient (qCO2) over time, re¯ecting adjustments by the microbial community to decreases in C availability in the casts of D. octaedra. While stabilization of soil carbon may be promoted by clay-associated carbohydrates which are more abundant in endogeic earthworm casts than in the surrounding soil (Shaw and Pawluk, 1986a,b) it is unlikely that epigeic earthworms would have a similar eect on C availability. Endogeic earthworms ingest highly decomposed amorphous organic matter associated with mineral material (Edwards and
M.A. McLean, D. Parkinson / Soil Biology & Biochemistry 32 (2000) 351±360
Bohlen, 1996). D. octaedra, like other epigeic earthworms, feeds on relatively undecomposed litter (Edwards and Bohlen, 1996) and epigeic gut passage results in comminuted but not chemically transformed organic materials (Ponge, 1991; Ziegler and Zech, 1992). In the pine forest in our study, it is clear that this species does mix organic and mineral material but this is not likely to result in the binding of carbon by clays as is found in endogeic earthworm casts. Changes in microbial activity observed by McLean and Parkinson (1997b) therefore may re¯ect stimulation of microbial growth as the microbial biomass is consumed by the earthworms or changes in fungal species composition. Earthworm activities can represent a major perturbation to litter and soil fungi and most fungal hyphal biomass is destroyed during gut passage through anecic and endogeic earthworms (e.g. Edwards and Bohlen, 1996) thus creating new substrates available for fungal colonization. Passage through endogeic and anecic earthworm guts signi®cantly alters fungal spore viability, decreasing the viability of many fungal species while increasing the viability of other fungal species (e.g. Edwards and Bohlen, 1996; Moody et al., 1996). If passage through epigeic earthworm guts has similar eects on hyphal and spore viability, dierential survival of spores able to colonize newly available substrates in earthworm casts may be an important mechanism in¯uencing the fungal community. Another consequence of the killing of fungal hyphal biomass during gut passage may be casts enriched in chitin, which is an important component in fungal cell walls (Cooke and Rayner, 1984). This might provide an ideal substrate for chitinolytic fungi such as species of the genera Trichoderma, Penicillium and Mortierella (Cooke and Rayner, 1984; Domsch et al., 1993) and may be a partial explanation for the observed positive association of T. koningii, T. polysporum and P. montanense with high worm activities in the FH layer. 3. Visser (1985); Brown (1995); Moody et al. (1995) and others have postulated that selective grazing is an important mechanism by which earthworms aect fungal communities. Although earthworms can select substrates inoculated with dierent species of fungi (e.g. Cooke, 1983; Moody et al., 1996), the large scale eects of this have not yet been demonstrated. The activities of D. octaedra at high densities and biomasses in the ®eld aected the fungal community by: (i) increasing fungal dominance, decreasing diversity and species richness in the FH layer and Ah and Bm horizons; (ii) dierentiat-
359
ing the fungal communities in the FH layer from those in the L layer and Bm horizons; (iii) increasing the similarity between the fungal communities in the FH layer and the Ah horizon; (iv) changing the fungal species composition.
Acknowledgements This work was supported by an NSERC Operating Grant to D.P. and by the Biodiversity Grants Program, through the joint eorts of the sportsmen of Alberta and the Alberta Department of Environmental Protection, Fish and Wildlife Trust Fund. Our thanks to D. Kolodka for technical assistance.
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